Goals: New treatments for liver diseases are needed. Groundbreaking advances in hepatology have set the stage for the development of treatments which directly inhibit disease associated RNAs, such as mRNAs over-expressed in hepatocellular carcinoma cells, mRNA of the PiZZ form of alpha-1 antitrypsin deficiency, and RNAs of liver pathogens. The proposed experiments will lead to new methods for eliminating specific pathogenic RNAs in the liver. Specific Aims: (A). Ribozymes cleave target RNAs specifically and therefore have recognized therapeutic potential. The delta hepatitis agent contains ribozymes which function effectively as self-cleavage structures in hepatocytes, but do not cleave intended target RNAs efficiently. To re-engineer these ribozymes, a recently developed in vitro selection method (Branch and Polaskova, 1995, NAR) and kinetic analysis will be sued to identify bonds which interfere with substrate turnover. By altering these bonds, superior therapeutic ribozymes for treating liver diseases will be developed. A derivative targeted against the conserved sequence element at the 3~ end of hepatitis C virus (HCV) RNA will be designed and tested. (B). RNA of the delta agent contains a novel element of local tertiary structure (Branch et. al. 1989, Science). This element is vulnerable to inhibition. Its structure and stability have been partially characterized (Branch et al., 1995, NAR) but need to be examined in greater detail so t hat specific inhibitors can be rationally designed. The minimum sequence and the ions needed to form and maintain this structure will be established by using standard techniques for RNA analysis, including non-denaturing gel electrophoresis. Conditions for obtaining homogenous preparations will be worked out, establishing the foundation for future high resolution structural determinations. (C) . Exposed nucleotides present in the core of the HCV 3' X tail of two different genotypes will be identified by partial nuclease digestion and other conventional methods of RNA structural analysis. A consensus structure will be sought. Chimeric ribozymes, with domains capable of recognizing exposed HCV nucleotides and nucleating ribozyme-HCV RNA binding, will be developed. Northern analysis of RNAs extracted from serum samples of patients will establish the size distribution of HCV RNAs containing the terminal sequence. These studies will lead to new treatments for liver diseases.